Research by genetic information analysis of various organisms has begun, and information on a number of genes including those of human and their base sequences, and on the proteins encoded by the gene sequences and sugar chains secondarily produced from these proteins, is being rapidly clarified. Functions of biopolymers such as genes, proteins and sugar chains whose sequences were clarified can be investigated by various methods. In terms of nucleic acids, major examples of the methods include Northern blotting and Southern blotting, which can be used for investigation of various genes in relation to expression of their biological functions by utilization of various nucleic acid-nucleic acid complementarities. In terms of proteins, examples of the methods include Western blotting, which can be used for investigation of functions and expression of proteins by utilization of protein-protein reactions.
In particular, in cases where a nucleic acid of interest (target nucleic acid) is to be detected in, for example, genetic diagnosis, identification of a pathogenic bacterium or detection of a single nucleotide polymorphism, a capture probe composed of nucleic acid is employed. In recent years, simultaneous detection of a plurality of types of target nucleic acids has been carried out using a DNA chip or DNA microarray, which contains a number of capture probes immobilized on a support. More specifically, the sequence of a target nucleic acid can be investigated by bringing a capture probe immobilized on a support into contact with the target nucleic acid, and then investigating the presence or absence of hybridization between the capture probe and the target nucleic acid to test their complementarity. Examples of common methods for the hybridization of the target nucleic acid include a method in which a labeling substance is introduced into the target nucleic acid, and the resulting nucleic acid is brought into contact with the capture probe, followed by detection of a signal from the labeling substance.
There are the following methods for the introduction of the labeling substance to the target nucleic acid: a method in which the labeling substance is introduced before the hybridization with the capture probe, and a method in which the labeling substance is introduced after the hybridization. The latter method is called the post-staining method. In this method, the target nucleic acid after hybridization is brought into contact with a labeling substance to introduce the labeling substance. This method allows use of a relatively large labeling substance since the labeling substance is bound after the hybridization. Moreover, the labeling step can be repeatedly carried out for enhancing the detected signal (Patent Documents 1 and 2). It is known that, in the labeling step of the post-staining method, a labeling solution containing a monovalent metal cation such as sodium ion at a concentration of as high as about 500 to 1000 mM is used.
On the other hand, since divalent metal cations are known to be substances that activate various nucleases, their positive use in methods for detection of nucleic acid using hybridization has been avoided, and there is no known technical idea in which a divalent metal cation is used in the labeling step of the post-staining method.